GEF-H1 modulates localized RhoA activation during cytokinesis under the control of mitotic kinases.

Formation of the mitotic cleavage furrow is dependent upon both microtubules and activity of the small GTPase RhoA. GEF-H1 is a microtubule-regulated exchange factor that couples microtubule dynamics to RhoA activation. GEF-H1 localized to the mitotic apparatus in HeLa cells, particularly at the tips of cortical microtubules and the midbody, and perturbation of GEF-H1 function induced mitotic aberrations, including asymmetric furrowing, membrane blebbing, and impaired cytokinesis. The mitotic kinases Aurora A/B and Cdk1/Cyclin B phosphorylate GEF-H1, thereby inhibiting GEF-H1 catalytic activity. Dephosphorylation of GEF-H1 occurs just prior to cytokinesis, accompanied by GEF-H1-dependent GTP loading on RhoA. Using a live cell biosensor, we demonstrate distinct roles for GEF-H1 and Ect2 in regulating Rho activity in the cleavage furrow, with GEF-H1 catalyzing Rho activation in response to Ect2-dependent localization and initiation of cell cleavage. Our results identify a GEF-H1-dependent mechanism to modulate localized RhoA activation during cytokinesis under the control of mitotic kinases.

Spatial and temporal analysis of Rac activation during live neutrophil chemotaxis.

The ability of cells to recognize and respond with directed motility to chemoattractant agents is critical to normal physiological function. Neutrophils represent the prototypic chemotactic cell in that they respond to signals initiated through the binding of bacterial peptides and other chemokines to G protein-coupled receptors with speeds of up to 30 microm/min. It has been hypothesized that localized regulation of cytoskeletal dynamics by Rho GTPases is critical to orchestrating cell movement. Using a FRET-based biosensor approach, we investigated the dynamics of Rac GTPase activation during chemotaxis of live primary human neutrophils. Rac has been implicated in establishing and maintaining the leading edge of motile cells, and we show that Rac is dynamically activated at specific locations in the extending leading edge. However, we also demonstrate activated Rac in the retracting tail of motile neutrophils. Rac activation is both stimulus and adhesion dependent. Expression of a dominant-negative Rac mutant confirms that Rac is functionally required both for tail retraction and for formation of the leading edge during chemotaxis. These data establish that Rac GTPase is spatially and temporally regulated to coordinate leading-edge extension and tail retraction during a complex motile response, the chemotaxis of human neutrophils.

The molecular basis for adhesion-mediated suppression of reactive oxygen species generation by human neutrophils.

Human neutrophil adherence to ECMs induces an initial inhibition of stimulated reactive oxygen species (ROS) formation, followed by an enhanced phase of oxidant production. The initial integrin-mediated suppression of ROS constitutes a mechanism to prevent inappropriate tissue damage as leukocytes migrate to inflammatory sites. The Rac2 guanosine 5'-triphosphatase (GTPase) is a critical regulatory component of the phagocyte NADPH oxidase. We show that activation of Rac2 is inhibited in adherent neutrophils, correlating with inhibition of ROS formation. Conversely, NADPH oxidase components p47 and p67 assemble normally, suggesting a specific action of adhesion on the Rac2 molecular switch. Reconstitution with activated Rac2 restored rapid NADPH oxidase activation kinetics to adherent neutrophils, establishing that inhibition was due to defective Rac2 activity. We provide evidence that integrins inhibit Rac2 activation via a membrane-associated guanine nucleotide exchange factor, likely to be Vav1. Activation of Vav1, but not its upstream activator, Syk, is suppressed by cell adhesion. Vav1 activity is inhibited due to dephosphorylation of the regulatory Tyr174 via enhanced tyrosine phosphatase activity in adherent cells. These studies identify an integrin-mediated pathway in which Vav1 is as a strong candidate for the critical regulatory point in suppression of Rac2 activation and ROS generation during inflammatory responses.

p21-activated kinase 1 phosphorylates and regulates 14-3-3 binding to GEF-H1, a microtubule-localized Rho exchange factor.

GEF-H1 is a guanine nucleotide exchange factor for Rho whose activity is regulated through a cycle of microtubule binding and release. Here we identify a region in the carboxyl terminus of GEF-H1 that is important for suppression of its guanine nucleotide exchange activity by microtubules. This portion of the protein includes a coiled-coil motif, a proline-rich motif that may interact with Src homology 3 domain-containing proteins, and a potential binding site for 14-3-3 proteins. We identify GEF-H1 as a binding target and substrate for p21-activated kinase 1 (PAK1), an effector of Rac and Cdc42 GTPases, using an affinity-based screen and localize a PAK1 phosphorylation site to the inhibitory carboxyl-terminal region of GEF-H1. We show that phosphorylation of GEF-H1 at Ser(885) by PAK1 induces 14-3-3 binding to the exchange factor and relocation of 14-3-3 to microtubules. Phosphorylation of GEF-H1 by PAK may be involved in regulation of GEF-H1 activity and may serve to coordinate Rho-, Rac-, and Cdc42-mediated signaling pathways.